1. Field of the Invention
This invention relates to methods and apparatus useful in analysis or testing of tissue samples.
2. Description of Related Art
The analysis of tissue is a valuable diagnostic tool used by pathologists to diagnose many illnesses and by medical researchers to obtain information about a cell structure.
In order to obtain information from a tissue sample usually it is necessary to perform a number of preliminary operations to prepare the sample for analysis. There are many variations of the procedures to prepare tissue samples for testing. These variations may be considered refinements to adapt the process for individual tissues or because a particular technique is better suited to identify a specific chemical substance or enzyme within the tissue sample. However the basic preparation techniques are essentially the same.
Typically such operations include processing of the tissue by fixation, dehydration, infiltration and embedding; mounting of the tissue on a slide and then staining the sample for analysis by a microscope.
Depending on the analysis or testing to be done, a sample may have to undergo a number of preliminary steps or treatments or procedures before it is ready to be analyzed for its informational content. Typically the procedures are complex and time consuming, involving many tightly sequenced steps often utilizing expensive and/or toxic materials.
These procedures usually must be performed in a critical order for each sample and each treatment often is time dependent. Additionally the laboratory often is under extreme pressure to perform many different analysis as soon as possible, entailing many different procedures and tests.
For example, a tissue sample may undergo an optical microscopic examination so that the relationship of various cells to each other may be determined or abnormalities may be uncovered. The tissue sample typically is an extremely thin strip of tissue so that light may be transmitted therethrough. The average thickness of the tissue sample or slice (often referred to as a section) is on the order of 2 to 8 microns. A relatively soft and pliable tissue such as might come from an organ of the human body, in its fresh state cannot be accurately cut into such thin sections. In addition, in order to see individual elements of the cells, such as the nucleus, the nucleolus, the cytoplasm and the cell membrane, it is preferable to color or stain them by different dyes to produce a contrasting appearance between the elements. Very limited dye staining can be done on fresh or recently living tissue without resorting to chemical processing. Typically a sample of tissue 2.0 to 2.5 square centimeters in area and 3 to 4 millimeters thick is utilized. The tissue sample is then fixed in a material (a fixative), which serves to preserve the cellular structure but also to stop further enzymic action, which could result in the putrification or autolysis of the tissue.
To prepare good samples for microscopic examination the initial step should kill the enzymic processes of the tissue and should alter or denature the proteins of the cell through fixation. The period of fixation may take several hours or even a few days depending upon the tissue type, sample size and type of fixative being used. After fixation, the tissue sample often is dehydrated by the removal of water from the sample through the use of increasing strengths of alcohol or of some other dehydrating fluid. Gradual dehydration is preferred because it causes less distortion to the sample than a rapid dehydration process.
The alcohol or dehydrating fluid is then replaced by a chemical, which can permeate the tissue sample and give it a consistency suitable for the preparation of thin sections without disintegration or splitting. Fat solvents, such as chloroform or toluene commonly are used for this step. The sample, which has been dehydrated by the infiltration of alcohol, is next exposed to several changes of solvent over a period that may last from a few hours to days until the alcohol is completely replaced by the solvent. The sample is then exposed to a molten wax. After the wax infiltration the sample is allowed to cool and the wax solidify so that the sample is entirely embedded in and infiltrated by the wax.
A microtome is then utilized to cut thin slices from the tissue sample. The slices are on the order of 5 to 6 microns thick. The cut thin sections are floated on water to spread or flatten the section. The section is then disposed on a glass slide, usually measuring about 8 by 2.5 millimeters.
The wax is then removed by exposing the sample to a solvent, the solvent removed by alcohol, and the alcohol removed by decreasing the alcoholic concentrations until eventually the tissue is once more infiltrated by water. The infiltration of the sample by water permits the staining of the cell constituents by water-soluble dyes.
Prior to the development of automated procedures for the preparation of tissue samples, it often took from 2 to 10 days for manual processing before a tissue could be examined under a microscope. In more recent years automated processes have been developed utilizing apparatus to transfer the sample from one fluid to another at defined intervals, and as a result tissue sample preparation time has been significantly reduced to between about 4 and 16 hours. Such automated apparatus are described in the patent literature (see for example Copeland et al. U.S. Pat. No. 5,595,707, Richards et al. U.S. Pat. No. 6,296,809 and Copeland et al. U.S. Pat. No. 6,352,861), and are available commercially such as the Benchmark® automated slide processor and the Discovery™ automated slide processor both available from Ventana Medical Systems, Inc. of Tucson, Ariz.
While automated slide processors have become widely adopted, heretofore automated processors could not be used when processing slides fixed with mercuric chloride (HgCl2) without taking the slides off-line to remove mercurous chloride precipitates.
Mercuric chloride is one of the more common and preferred fixatives. Mercuric chloride is a powerful protein precipitant and forms intermolecular mercury links between S—H carboxyl and amino groups. It penetrates reasonably well, shrinks tissue less than many other common protein coagulants, hardens tissue moderately, and distorts the cells less than many other common fixatives and thus is preferred in many applications. Notwithstanding the foregoing, a primary disadvantage to the use of mercuric chloride based fixatives is the formation of precipitates on the tissue section. The precipitates, which are formed as crystalline granules, comprise mercurous chloride (HgCl), which are highly conspicuous and interfere with reading of a stain at the microscopic level. (See FIG. 1).
While slides fixed with other fixatives, such as formaldehyde, formalin, picric acid, etc., have been successfully processed on automated systems as above described, for slides fixed with mercuric chloride, two additional manual steps are required in order to remove the mercurous chloride precipitates as follows:
(1) The slides first are placed into an aqueous solution that contains iodine. This step converts the mercurous chloride precipitates to water soluble mercuric chloride, and mercuric iodine according to the reaction:2HgCl+I2=HgCl2+HgI2 
(2) The slides are then rinsed and placed in an aqueous solution containing sodium thiosulfate to convert any free iodide to water soluble sodium iodide according to the reaction:2NA2S2O3+I2=2NaI+Na2S4O6 While these added process steps remove the mercurous chloride precipitates (see FIG. 2), these two added process steps, which typically are run off line, add significant costs and delays.